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1.
Protein Sci ; 31(9): e4409, 2022 09.
Article in English | MEDLINE | ID: covidwho-2003635

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid protein is an essential structural component of mature virions, encapsulating the genomic RNA and modulating RNA transcription and replication. Several of its activities might be associated with the protein's ability to undergo liquid-liquid phase separation. NSARS-CoV-2 contains an intrinsically disordered region at its N-terminus (NTE) that can be phosphorylated and is affected by mutations found in human COVID-19 infections, including in the Omicron variant of concern. Here, we show that NTE deletion decreases the range of RNA concentrations that can induce phase separation of NSARS-CoV-2 . In addition, deletion of the prion-like NTE allows NSARS-CoV-2 droplets to retain their liquid-like nature during incubation. We further demonstrate that RNA-binding engages multiple parts of the NTE and changes NTE's structural properties. The results form the foundation to characterize the impact of N-terminal mutations and post-translational modifications on the molecular properties of the SARS-CoV-2 nucleocapsid protein. STATEMENT: The nucleocapsid protein of SARS-CoV-2 plays an important role in both genome packaging and viral replication upon host infection. Replication has been associated with RNA-induced liquid-liquid phase separation of the nucleocapsid protein. We present insights into the role of the N-terminal part of the nucleocapsid protein in the protein's RNA-mediated liquid-liquid phase separation.


Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19/genetics , Humans , Nucleocapsid Proteins/chemistry , Nucleocapsid Proteins/genetics , Nucleocapsid Proteins/metabolism , RNA, Viral/chemistry , SARS-CoV-2/genetics
2.
Viruses ; 14(8)2022 08 16.
Article in English | MEDLINE | ID: covidwho-1988001

ABSTRACT

Most pandemics of recent decades can be traced to RNA viruses, including HIV, SARS, influenza, dengue, Zika, and SARS-CoV-2. These RNA viruses impose considerable social and economic burdens on our society, resulting in a high number of deaths and high treatment costs. As these RNA viruses utilize an RNA genome, which is important for different stages of the viral life cycle, including replication, translation, and packaging, studying how the genome folds is important to understand virus function. In this review, we summarize recent advances in computational and high-throughput RNA structure-mapping approaches and their use in understanding structures within RNA virus genomes. In particular, we focus on the genome structures of the dengue, Zika, and SARS-CoV-2 viruses due to recent significant outbreaks of these viruses around the world.


Subject(s)
COVID-19 , Dengue , RNA Viruses , Zika Virus Infection , Zika Virus , Dengue/genetics , Genome, Viral , Humans , RNA , RNA Viruses/genetics , RNA, Viral/chemistry , RNA, Viral/genetics , SARS-CoV-2/genetics , Zika Virus/genetics , Zika Virus Infection/genetics
3.
Nature ; 609(7928): 793-800, 2022 09.
Article in English | MEDLINE | ID: covidwho-1984402

ABSTRACT

The RNA genome of SARS-CoV-2 contains a 5' cap that facilitates the translation of viral proteins, protection from exonucleases and evasion of the host immune response1-4. How this cap is made in SARS-CoV-2 is not completely understood. Here we reconstitute the N7- and 2'-O-methylated SARS-CoV-2 RNA cap (7MeGpppA2'-O-Me) using virally encoded non-structural proteins (nsps). We show that the kinase-like nidovirus RdRp-associated nucleotidyltransferase (NiRAN) domain5 of nsp12 transfers the RNA to the amino terminus of nsp9, forming a covalent RNA-protein intermediate (a process termed RNAylation). Subsequently, the NiRAN domain transfers the RNA to GDP, forming the core cap structure GpppA-RNA. The nsp146 and nsp167 methyltransferases then add methyl groups to form functional cap structures. Structural analyses of the replication-transcription complex bound to nsp9 identified key interactions that mediate the capping reaction. Furthermore, we demonstrate in a reverse genetics system8 that the N terminus of nsp9 and the kinase-like active-site residues in the NiRAN domain are required for successful SARS-CoV-2 replication. Collectively, our results reveal an unconventional mechanism by which SARS-CoV-2 caps its RNA genome, thus exposing a new target in the development of antivirals to treat COVID-19.


Subject(s)
RNA Caps , RNA, Viral , SARS-CoV-2 , Viral Proteins , Antiviral Agents , COVID-19/drug therapy , COVID-19/virology , Catalytic Domain , Guanosine Diphosphate/metabolism , Humans , Methyltransferases/metabolism , Nucleotidyltransferases/chemistry , Nucleotidyltransferases/metabolism , Protein Domains , RNA Caps/chemistry , RNA Caps/genetics , RNA Caps/metabolism , RNA, Viral/chemistry , RNA, Viral/genetics , RNA, Viral/metabolism , RNA-Dependent RNA Polymerase/metabolism , SARS-CoV-2/enzymology , SARS-CoV-2/genetics , SARS-CoV-2/metabolism , Viral Proteins/chemistry , Viral Proteins/metabolism
5.
J Phys Chem Lett ; 13(31): 7197-7205, 2022 Aug 11.
Article in English | MEDLINE | ID: covidwho-1972509

ABSTRACT

Remdesivir is one nucleotide analogue prodrug capable to terminate RNA synthesis in SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) by two distinct mechanisms. Although the "delayed chain termination" mechanism has been extensively investigated, the "template-dependent" inhibitory mechanism remains elusive. In this study, we have demonstrated that remdesivir embedded in the template strand seldom directly disrupted the complementary NTP incorporation at the active site. Instead, the translocation of remdesivir from the +2 to the +1 site was hindered due to the steric clash with V557. Moreover, we have elucidated the molecular mechanism characterizing the drug resistance upon V557L mutation. Overall, our studies have provided valuable insight into the "template-dependent" inhibitory mechanism exerted by remdesivir on SARS-CoV-2 RdRp and paved venues for an alternative antiviral strategy for the COVID-19 pandemic. As the "template-dependent" inhibition occurs across diverse viral RdRps, our findings may also shed light on a common acting mechanism of inhibitors.


Subject(s)
COVID-19 , SARS-CoV-2 , Antiviral Agents/chemistry , COVID-19/drug therapy , Humans , Pandemics , RNA, Viral/chemistry , RNA-Dependent RNA Polymerase , Viral Transcription
6.
Biochem Biophys Res Commun ; 625: 53-59, 2022 10 15.
Article in English | MEDLINE | ID: covidwho-1966378

ABSTRACT

The novel Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2 or COVID-19) has caused a global pandemic. The SARS-CoV-2 RNA genome is replicated by a conserved "core" replication-transcription complex (RTC) containing an error-prone RNA-dependent RNA polymerase holoenzyme (holo-RdRp, nsp12-nsp7-nsp8) and a RNA proofreading nuclease (nsp14-nsp10). Although structures and functions of SARS-CoV-2 holo-RdRp have been extensively studied and ribonucleotide-analog inhibitors, such as Remdesivir, have been treated for COVID-19 patients, the substrate and nucleotide specificity of SARS-CoV-2 holo-RdRp remain unknown. Here, our biochemical analysis of SARS-CoV-2 holo-RdRp reveals that it has a robust DNA-dependent RNA polymerase activity, in addition to its intrinsic RNA-dependent RNA polymerase activity. Strikingly, SARS-CoV-2 holo-RdRp fully extends RNAs with a low-fidelity even when only ATP and pyrimidine nucleotides, in particular CTP, are provided. This ATP-dependent error-prone ribonucleotide incorporation by SARS-CoV-2 holo-RdRp resists excision by the RNA proofreading nuclease in vitro. Our collective results suggest that a physiological concentration of ATP likely contributes to promoting the error-prone incorporation of ribonucleotides and ribonucleotide-analogs by SARS-CoV-2 holo-RdRp and provide a useful foundation to develop ribonucleotide analogs as an effective therapeutic strategy to combat coronavirus-mediated outbreak.


Subject(s)
COVID-19 , SARS-CoV-2 , Adenosine Triphosphate , Antiviral Agents/chemistry , DNA-Directed RNA Polymerases , Humans , RNA, Viral/chemistry , RNA, Viral/genetics , RNA-Dependent RNA Polymerase , Ribonucleotides , SARS-CoV-2/genetics , Viral Nonstructural Proteins/chemistry
7.
Nat Commun ; 13(1): 4284, 2022 07 25.
Article in English | MEDLINE | ID: covidwho-1956403

ABSTRACT

The SARS-CoV-2 frameshifting element (FSE), a highly conserved mRNA region required for correct translation of viral polyproteins, defines an excellent therapeutic target against Covid-19. As discovered by our prior graph-theory analysis with SHAPE experiments, the FSE adopts a heterogeneous, length-dependent conformational landscape consisting of an assumed 3-stem H-type pseudoknot (graph motif 3_6), and two alternative motifs (3_3 and 3_5). Here, for the first time, we build and simulate, by microsecond molecular dynamics, 30 models for all three motifs plus motif-stabilizing mutants at different lengths. Our 3_6 pseudoknot systems, which agree with experimental structures, reveal interconvertible L and linear conformations likely related to ribosomal pausing and frameshifting. The 3_6 mutant inhibits this transformation and could hamper frameshifting. Our 3_3 systems exhibit length-dependent stem interactions that point to a potential transition pathway connecting the three motifs during ribosomal elongation. Together, our observations provide new insights into frameshifting mechanisms and anti-viral strategies.


Subject(s)
COVID-19 , Frameshifting, Ribosomal , Base Sequence , Humans , Nucleic Acid Conformation , RNA, Viral/chemistry , RNA, Viral/genetics , SARS-CoV-2/genetics
8.
PLoS One ; 17(3): e0264855, 2022.
Article in English | MEDLINE | ID: covidwho-1896450

ABSTRACT

Since December 2019 the world has been facing the outbreak of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Identification of infected patients and discrimination from other respiratory infections have so far been accomplished by using highly specific real-time PCRs. Here we present a rapid multiplex approach (RespiCoV), combining highly multiplexed PCRs and MinION sequencing suitable for the simultaneous screening for 41 viral and five bacterial agents related to respiratory tract infections, including the human coronaviruses NL63, HKU1, OC43, 229E, Middle East respiratory syndrome coronavirus, SARS-CoV, and SARS-CoV-2. RespiCoV was applied to 150 patient samples with suspected SARS-CoV-2 infection and compared with specific real-time PCR. Additionally, several respiratory tract pathogens were identified in samples tested positive or negative for SARS-CoV-2. Finally, RespiCoV was experimentally compared to the commercial RespiFinder 2SMART multiplex screening assay (PathoFinder, The Netherlands).


Subject(s)
Bacteria/genetics , COVID-19/diagnosis , High-Throughput Nucleotide Sequencing/methods , RNA Viruses/genetics , Respiratory Tract Infections/diagnosis , SARS-CoV-2/genetics , Bacteria/isolation & purification , COVID-19/virology , Coronavirus/genetics , Coronavirus/isolation & purification , DNA, Bacterial/chemistry , DNA, Bacterial/metabolism , Herpesvirus 1, Human/genetics , Herpesvirus 1, Human/isolation & purification , Humans , Multiplex Polymerase Chain Reaction , Nanopores , Orthomyxoviridae/genetics , Orthomyxoviridae/isolation & purification , RNA Viruses/isolation & purification , RNA, Viral/chemistry , RNA, Viral/metabolism , Respiratory Tract Infections/microbiology , Respiratory Tract Infections/virology , SARS-CoV-2/isolation & purification
9.
J Virol ; 96(8): e0194621, 2022 04 27.
Article in English | MEDLINE | ID: covidwho-1861580

ABSTRACT

Hepatitis C virus (HCV) is a positive-strand RNA virus that remains one of the main contributors to chronic liver disease worldwide. Studies over the last 30 years have demonstrated that HCV contains a highly structured RNA genome and many of these structures play essential roles in the HCV life cycle. Despite the importance of riboregulation in this virus, most of the HCV RNA genome remains functionally unstudied. Here, we report a complete secondary structure map of the HCV RNA genome in vivo, which was studied in parallel with the secondary structure of the same RNA obtained in vitro. Our results show that HCV is folded extensively in the cellular context. By performing comprehensive structural analyses on both in vivo data and in vitro data, we identify compact and conserved secondary and tertiary structures throughout the genome. Genetic and evolutionary functional analyses demonstrate that many of these elements play important roles in the virus life cycle. In addition to providing a comprehensive map of RNA structures and riboregulatory elements in HCV, this work provides a resource for future studies aimed at identifying therapeutic targets and conducting further mechanistic studies on this important human pathogen. IMPORTANCE HCV has one of the most highly structured RNA genomes studied to date, and it is a valuable model system for studying the role of RNA structure in protein-coding genes. While previous studies have identified individual cases of regulatory RNA structures within the HCV genome, the full-length structure of the HCV genome has not been determined in vivo. Here, we present the complete secondary structure map of HCV determined both in cells and from corresponding transcripts generated in vitro. In addition to providing a comprehensive atlas of functional secondary structural elements throughout the genomic RNA, we identified a novel set of tertiary interactions and demonstrated their functional importance. In terms of broader implications, the pipeline developed in this study can be applied to other long RNAs, such as long noncoding RNAs. In addition, the RNA structural motifs characterized in this study broaden the repertoire of known riboregulatory elements.


Subject(s)
Genome, Viral , Hepacivirus , RNA, Viral , Genome, Viral/genetics , Hepacivirus/genetics , Hepatitis C/virology , Humans , RNA, Untranslated/chemistry , RNA, Viral/chemistry , RNA, Viral/genetics
10.
Sci Rep ; 12(1): 3860, 2022 03 09.
Article in English | MEDLINE | ID: covidwho-1799576

ABSTRACT

Non-structural protein 15 (Nsp15) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) forms a homo hexamer and functions as an endoribonuclease. Here, we propose that Nsp15 activity may be inhibited by preventing its hexamerization through drug binding. We first explored the stable conformation of the Nsp15 monomer as the global free energy minimum conformation in the free energy landscape using a combination of parallel cascade selection molecular dynamics (PaCS-MD) and the Markov state model (MSM), and found that the Nsp15 monomer forms a more open conformation with larger druggable pockets on the surface. Targeting the pockets with high druggability scores, we conducted ligand docking and identified compounds that tightly bind to the Nsp15 monomer. The top poses with Nsp15 were subjected to binding free energy calculations by dissociation PaCS-MD and MSM (dPaCS-MD/MSM), indicating the stability of the complexes. One of the identified pockets, which is distinctively bound by inosine analogues, may be an alternative binding site to stabilize viral RNA binding and/or an alternative catalytic site. We constructed a stable RNA structure model bound to both UTP and alternative binding sites, providing a reasonable proposed model of the Nsp15/RNA complex.


Subject(s)
Endoribonucleases/metabolism , RNA, Viral/chemistry , SARS-CoV-2/metabolism , Viral Nonstructural Proteins/metabolism , Antiviral Agents/chemistry , Antiviral Agents/metabolism , Binding Sites , COVID-19/pathology , COVID-19/virology , Endoribonucleases/antagonists & inhibitors , Humans , Markov Chains , Molecular Docking Simulation , Molecular Dynamics Simulation , Nucleic Acid Conformation , Protein Multimerization , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Static Electricity , Viral Nonstructural Proteins/antagonists & inhibitors
11.
Radiat Res ; 198(1): 68-80, 2022 07 01.
Article in English | MEDLINE | ID: covidwho-1793416

ABSTRACT

Here we show an interplay between the structures present in ionization tracks and nucleocapsid RNA structural biology, using fast ion-beam inactivation of the severe acute respiratory syndrome coronavirus (SARS-CoV) virion as an example. This interplay could be a key factor in predicting dose-inactivation curves for high-energy ion-beam inactivation of virions. We also investigate the adaptation of well-established cross-section data derived from radiation interactions with water to the interactions involving the components of a virion, going beyond the density-scaling approximation developed previously. We conclude that solving one of the grand challenges of structural biology - the determination of RNA tertiary/quaternary structure - is linked to predicting ion-beam inactivation of viruses and that the two problems can be mutually informative. Indeed, our simulations show that fast ion beams have a key role to play in elucidating RNA tertiary/quaternary structure.


Subject(s)
Nucleic Acid Conformation , RNA, Viral/chemistry , SARS-CoV-2 , Virus Inactivation , Ions , Models, Molecular , RNA, Viral/metabolism , Radiobiology/methods , SARS-CoV-2/chemistry , Viral Proteins/chemistry , Viral Proteins/metabolism , Virion/chemistry
12.
Immunogenetics ; 74(5): 455-463, 2022 10.
Article in English | MEDLINE | ID: covidwho-1750684

ABSTRACT

G-quadruplex structure or Putative Quadruplex Sequences (PQSs) are abundant in human, microbial, DNA, or RNA viral genomes. These sequences in RNA viral genome play critical roles in integration into human genome as LTR (Long Terminal Repeat), genome replication, chromatin rearrangements, gene regulation, antigen variation (Av), and virulence. Here, we investigated whether the genome of SARS-CoV2, an RNA virus, contained such potential G-quadruplex structures. Using bioinformatic tools, we searched for such sequences and found thirty-seven (forward strand (twenty-five) + reverse strand (Twelve)) QGRSs (Quadruplex forming G-Rich Sequences)/PQSs in SARS-CoV2 genome. These sequences are dispersed mainly in the upstream of SARS-CoV2 genes. We discuss whether existing PQS/QGRS ligands could inhibit the SARS-CoV2 replication and gene transcription as has been observed in other RNA viruses. Further experimental validation would determine the role of these G-quadruplex sequences in SARS-CoV2 genome function to survive in the host cells and identify therapeutic agents to destabilize these PQSs/QGRSs.


Subject(s)
COVID-19 , G-Quadruplexes , COVID-19/genetics , DNA , Humans , RNA, Viral/chemistry , RNA, Viral/genetics , SARS-CoV-2/genetics
13.
EBioMedicine ; 76: 103861, 2022 Feb.
Article in English | MEDLINE | ID: covidwho-1734342

ABSTRACT

BACKGROUND: Since late 2019, SARS-CoV-2 infection has resulted in COVID-19 accompanied by diverse clinical manifestations. However, the underlying mechanism of how SARS-CoV-2 interacts with host and develops multiple symptoms is largely unexplored. METHODS: Bioinformatics analysis determined the sequence similarity between SARS-CoV-2 and human genomes. Diverse fragments of SARS-CoV-2 genome containing Human Identical Sequences (HIS) were cloned into the lentiviral vector. HEK293T, MRC5 and HUVEC were infected with laboratory-packaged lentivirus or transfected with plasmids or antagomirs for HIS. Quantitative RT-PCR and chromatin immunoprecipitation assay detected gene expression and H3K27ac enrichment, respectively. UV-Vis spectroscopy assessed the interaction between HIS and their target locus. Enzyme-linked immunosorbent assay evaluated the hyaluronan (HA) levels of culture supernatant and plasma of COVID-19 patients. FINDINGS: Five short sequences (24-27 nt length) sharing identity between SARS-CoV-2 and human genome were identified. These RNA elements were highly conserved in primates. The genomic fragments containing HIS were predicted to form hairpin structures in silico similar to miRNA precursors. HIS may function through direct genomic interaction leading to activation of host enhancers, and upregulation of adjacent and distant genes, including cytokine genes and hyaluronan synthase 2 (HAS2). HIS antagomirs and Cas13d-mediated HIS degradation reduced HAS2 expression. Severe COVID-19 patients displayed decreased lymphocytes and elevated D-dimer, and C-reactive proteins, as well as increased plasma hyaluronan. Hymecromone inhibited hyaluronan production in vitro, and thus could be further investigated as a therapeutic option for preventing severe outcome in COVID-19 patients. INTERPRETATION: HIS of SARS-CoV-2 could promote COVID-19 progression by upregulating hyaluronan, providing novel targets for treatment. FUNDING: The National Key R&D Program of China (2018YFC1005004), Major Special Projects of Basic Research of Shanghai Science and Technology Commission (18JC1411101), and the National Natural Science Foundation of China (31872814, 32000505).


Subject(s)
Gene Regulatory Networks/genetics , Genome, Human , Hyaluronic Acid/metabolism , RNA, Viral/genetics , SARS-CoV-2/genetics , Antagomirs/metabolism , Argonaute Proteins/genetics , Base Sequence , COVID-19/pathology , COVID-19/virology , Cell Line , Disease Progression , Enhancer Elements, Genetic/genetics , Humans , Hyaluronan Synthases/genetics , Hyaluronan Synthases/metabolism , Hyaluronic Acid/blood , MicroRNAs/genetics , RNA, Viral/chemistry , SARS-CoV-2/isolation & purification , SARS-CoV-2/pathogenicity , Up-Regulation
14.
RNA ; 28(5): 729-741, 2022 05.
Article in English | MEDLINE | ID: covidwho-1724733

ABSTRACT

The 5'UTR part of coronavirus genomes plays key roles in the viral replication cycle and translation of viral mRNAs. The first 75-80 nt, also called the leader sequence, are identical for genomic mRNA and subgenomic mRNAs. Recently, it was shown that cooperative actions of a 5'UTR segment and the nonstructural protein NSP1 are essential for both the inhibition of host mRNAs and for specific translation of viral mRNAs. Here, sequence analyses of both the 5'UTR RNA segment and the NSP1 protein have been done for several coronaviruses, with special attention to the betacoronaviruses. The conclusions are: (i) precise specific molecular signatures can be found in both the RNA and the NSP1 protein; (ii) both types of signatures correlate between each other. Indeed, definite sequence motifs in the RNA correlate with sequence motifs in the protein, indicating a coevolution between the 5'UTR and NSP1 in betacoronaviruses. Experimental mutational data on 5'UTR and NSP1 from SARS-CoV-2 using cell-free translation extracts support these conclusions and show that some conserved key residues in the amino-terminal half of the NSP1 protein are essential for evasion to the inhibitory effect of NSP1 on translation.


Subject(s)
COVID-19 , RNA, Viral , SARS-CoV-2 , Viral Nonstructural Proteins , 5' Untranslated Regions , COVID-19/virology , Humans , Protein Biosynthesis/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Viral/chemistry , SARS-CoV-2/genetics , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism
15.
Nat Commun ; 13(1): 1128, 2022 03 02.
Article in English | MEDLINE | ID: covidwho-1721520

ABSTRACT

SARS-CoV-2 is a betacoronavirus with a single-stranded, positive-sense, 30-kilobase RNA genome responsible for the ongoing COVID-19 pandemic. Although population average structure models of the genome were recently reported, there is little experimental data on native structural ensembles, and most structures lack functional characterization. Here we report secondary structure heterogeneity of the entire SARS-CoV-2 genome in two lines of infected cells at single nucleotide resolution. Our results reveal alternative RNA conformations across the genome and at the critical frameshifting stimulation element (FSE) that are drastically different from prevailing population average models. Importantly, we find that this structural ensemble promotes frameshifting rates much higher than the canonical minimal FSE and similar to ribosome profiling studies. Our results highlight the value of studying RNA in its full length and cellular context. The genomic structures detailed here lay groundwork for coronavirus RNA biology and will guide the design of SARS-CoV-2 RNA-based therapeutics.


Subject(s)
COVID-19/virology , RNA, Viral/chemistry , SARS-CoV-2/genetics , Frameshifting, Ribosomal , Genome, Viral , Humans , Nucleic Acid Conformation , RNA, Viral/genetics , RNA, Viral/metabolism , SARS-CoV-2/chemistry , SARS-CoV-2/metabolism
16.
Brief Bioinform ; 23(3)2022 05 13.
Article in English | MEDLINE | ID: covidwho-1713563

ABSTRACT

SARS-CoV-2 is a novel positive-sense single-stranded RNA virus from the Coronaviridae family (genus Betacoronavirus), which has been established as causing the COVID-19 pandemic. The genome of SARS-CoV-2 is one of the largest among known RNA viruses, comprising of at least 26 known protein-coding loci. Studies thus far have outlined the coding capacity of the positive-sense strand of the SARS-CoV-2 genome, which can be used directly for protein translation. However, it has been recently shown that transcribed negative-sense viral RNA intermediates that arise during viral genome replication from positive-sense viruses can also code for proteins. No studies have yet explored the potential for negative-sense SARS-CoV-2 RNA intermediates to contain protein-coding loci. Thus, using sequence and structure-based bioinformatics methodologies, we have investigated the presence and validity of putative negative-sense ORFs (nsORFs) in the SARS-CoV-2 genome. Nine nsORFs were discovered to contain strong eukaryotic translation initiation signals and high codon adaptability scores, and several of the nsORFs were predicted to interact with RNA-binding proteins. Evolutionary conservation analyses indicated that some of the nsORFs are deeply conserved among related coronaviruses. Three-dimensional protein modeling revealed the presence of higher order folding among all putative SARS-CoV-2 nsORFs, and subsequent structural mimicry analyses suggest similarity of the nsORFs to DNA/RNA-binding proteins and proteins involved in immune signaling pathways. Altogether, these results suggest the potential existence of still undescribed SARS-CoV-2 proteins, which may play an important role in the viral lifecycle and COVID-19 pathogenesis.


Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19/genetics , Genome, Viral , Humans , Pandemics , RNA, Viral/chemistry , RNA, Viral/genetics , RNA-Binding Proteins/genetics , SARS-CoV-2/genetics
17.
Nat Commun ; 13(1): 988, 2022 02 21.
Article in English | MEDLINE | ID: covidwho-1713165

ABSTRACT

Translating ribosomes unwind mRNA secondary structures by three basepairs each elongation cycle. Despite the ribosome helicase, certain mRNA stem-loops stimulate programmed ribosomal frameshift by inhibiting translation elongation. Here, using mutagenesis, biochemical and single-molecule experiments, we examine whether high stability of three basepairs, which are unwound by the translating ribosome, is critical for inducing ribosome pauses. We find that encountering frameshift-inducing mRNA stem-loops from the E. coli dnaX mRNA and the gag-pol transcript of Human Immunodeficiency Virus (HIV) hinders A-site tRNA binding and slows down ribosome translocation by 15-20 folds. By contrast, unwinding of first three basepairs adjacent to the mRNA entry channel slows down the translating ribosome by only 2-3 folds. Rather than high thermodynamic stability, specific length and structure enable regulatory mRNA stem-loops to stall translation by forming inhibitory interactions with the ribosome. Our data provide the basis for rationalizing transcriptome-wide studies of translation and searching for novel regulatory mRNA stem-loops.


Subject(s)
Frameshifting, Ribosomal , RNA, Messenger/chemistry , Bacterial Proteins/genetics , DNA Polymerase III/genetics , Escherichia coli/genetics , Fluorescence Resonance Energy Transfer , HIV/genetics , Nucleic Acid Conformation , RNA, Bacterial/chemistry , RNA, Bacterial/metabolism , RNA, Messenger/metabolism , RNA, Transfer/metabolism , RNA, Viral/chemistry , RNA, Viral/metabolism , Single Molecule Imaging , Thermodynamics
18.
Nat Commun ; 13(1): 968, 2022 02 18.
Article in English | MEDLINE | ID: covidwho-1705624

ABSTRACT

DNA/RNA-gold nanoparticle (DNA/RNA-AuNP) nanoprobes have been widely employed for nanobiotechnology applications. Here, we discover that both thiolated and non-thiolated DNA/RNA can be efficiently attached to AuNPs to achieve high-stable spherical nucleic acid (SNA) within minutes under a domestic microwave (MW)-assisted heating-dry circumstance. Further studies show that for non-thiolated DNA/RNA the conjugation is poly (T/U) tag dependent. Spectroscopy, test strip hybridization, and loading counting experiments indicate that low-affinity poly (T/U) tag mediates the formation of a standing-up conformation, which is distributed in the outer layer of SNA structure. In further application studies, CRISPR/Cas9-sgRNA (136 bp), SARS-CoV-2 RNA fragment (1278 bp), and rolling circle amplification (RCA) DNA products (over 1000 bp) can be successfully attached on AuNPs, which overcomes the routine methods in long-chain nucleic acid-AuNP conjugation, exhibiting great promise in biosensing and nucleic acids delivery applications. Current heating-dry strategy has improved traditional DNA/RNA-AuNP conjugation methods in simplicity, rapidity, cost, and universality.


Subject(s)
Biosensing Techniques/methods , Gold/chemistry , Metal Nanoparticles/chemistry , Biotechnology/methods , COVID-19/diagnosis , COVID-19/virology , COVID-19 Nucleic Acid Testing/methods , DNA/chemistry , Heating/methods , Humans , Limit of Detection , Microwaves , Nanomedicine/methods , RNA, Viral/chemistry , RNA, Viral/genetics , RNA, Viral/isolation & purification , SARS-CoV-2/genetics
19.
Int J Mol Sci ; 23(5)2022 Feb 23.
Article in English | MEDLINE | ID: covidwho-1700574

ABSTRACT

Influenza A virus (IAV) is a member of the single-stranded RNA (ssRNA) family of viruses. The most recent global pandemic caused by the SARS-CoV-2 virus has shown the major threat that RNA viruses can pose to humanity. In comparison, influenza has an even higher pandemic potential as a result of its high rate of mutations within its relatively short (<13 kbp) genome, as well as its capability to undergo genetic reassortment. In light of this threat, and the fact that RNA structure is connected to a broad range of known biological functions, deeper investigation of viral RNA (vRNA) structures is of high interest. Here, for the first time, we propose a secondary structure for segment 8 vRNA (vRNA8) of A/California/04/2009 (H1N1) formed in the presence of cellular and viral components. This structure shows similarities with prior in vitro experiments. Additionally, we determined the location of several well-defined, conserved structural motifs of vRNA8 within IAV strains with possible functionality. These RNA motifs appear to fold independently of regional nucleoprotein (NP)-binding affinity, but a low or uneven distribution of NP in each motif region is noted. This research also highlights several accessible sites for oligonucleotide tools and small molecules in vRNA8 in a cellular environment that might be a target for influenza A virus inhibition on the RNA level.


Subject(s)
Gene Expression Regulation, Viral , Genome, Viral/genetics , Influenza A Virus, H1N1 Subtype/genetics , Nucleic Acid Conformation , RNA, Viral/chemistry , Animals , Base Sequence , Dogs , Humans , Influenza A Virus, H1N1 Subtype/metabolism , Influenza, Human/virology , Madin Darby Canine Kidney Cells , Models, Molecular , Nucleotide Motifs/genetics , RNA Folding , RNA, Viral/genetics , Viral Proteins/genetics , Viral Proteins/metabolism
20.
Sci Rep ; 12(1): 2533, 2022 02 15.
Article in English | MEDLINE | ID: covidwho-1692569

ABSTRACT

To evaluate the incidence of COVID-19 infection in health care workers from the start of the COVID-19 pandemic in NE Italy, vaccination with BNT162b2. This was a retrospective cohort study. Healthcare workers were routinely tested for SARS-CoV-2 infection using real-time polymerase chain reaction tests in nasopharyngeal swabs. Logistic regression was used to calculate the incidence rate ratios (IRRs) of the factors associated with COVID-19. A total of 4251 workers were followed up, and the prevalence of COVID-19 was 13.6%. In March 2021 the incidence of infection was 4.88 and 103.55 cases for 100,000 person-days in vaccinated and non-vaccinated workers, respectively, with an adjusted IRRs of 0.05 (95% CI 0.02-0.08). Our study evaluated the monthly incidence in health care workers in Trieste hospitals before and after vaccination, finding an estimated vaccine effectiveness of 95% in health care workers routinely tested.


Subject(s)
/administration & dosage , COVID-19/epidemiology , Health Personnel/statistics & numerical data , Adult , COVID-19/prevention & control , COVID-19/virology , Clinical Trials as Topic , Female , Follow-Up Studies , Humans , Incidence , Logistic Models , Male , Middle Aged , Nasopharynx/virology , RNA, Viral/chemistry , RNA, Viral/metabolism , Retrospective Studies , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Vaccination
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